Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1994 Apr;60(4):1335–1341. doi: 10.1128/aem.60.4.1335-1341.1994

Molecular characterization of Frankia microsymbionts from spore-positive and spore-negative nodules in a natural alder stand.

P Simonet 1, M Bosco 1, C Chapelon 1, A Moiroud 1, P Normand 1
PMCID: PMC201478  PMID: 8017920

Abstract

Among the Frankia strains capable of establishing a nitrogen-fixing symbiosis with actinorrhizal plants, in planta sporangial formation is not universal and has led to the distinction between spore-positive (Sp+) and spore-negative (Sp-) nodules. Numerous Frankia strains have been isolated in pure culture from Sp+ nodules of different host plants, but, although they were able to reinfect their respective host plant, none of them was able to differentiate endophytic sporangia under laboratory conditions. The first step of this study was to demonstrate, at the molecular level, the existence of specific Sp+ strain genotypes differing from Sp- strain genotypes in a single alder stand. In a second step, by way of PCR amplification and sequencing of the PCR products, we have characterized oligonucleotide primers specific for the genus Frankia and for each of the two types of Frankia microsymbionts able, or not, to differentiate sporangia inside natural green alder nodules. These primers applied in PCRs with DNA extracted from nodules confirmed the morphological identification and revealed the presence of nodules colonized by both types of actinomycetes. Finally, a preliminary PCR study was conducted with DNA extracted directly from soil samples which permitted checking the rhizosphere of Sp+ and Sp- nodules for the presence of the corresponding strains.

Full text

PDF
1335

Images in this article

1337Image
on p.1337
1338Image
on p.1338
1340Image
on p.1340

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bosco M., Fernandez M. P., Simonet P., Materassi R., Normand P. Evidence that some Frankia sp. strains are able to cross boundaries between Alnus and Elaeagnus host specificity groups. Appl Environ Microbiol. 1992 May;58(5):1569–1576. doi: 10.1128/aem.58.5.1569-1576.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Callaham D., Deltredici P., Torrey J. G. Isolation and Cultivation in vitro of the Actinomycete Causing Root Nodulation in Comptonia. Science. 1978 Feb 24;199(4331):899–902. doi: 10.1126/science.199.4331.899. [DOI] [PubMed] [Google Scholar]
  3. Cournoyer B., Gouy M., Normand P. Molecular phylogeny of the symbiotic actinomycetes of the genus Frankia matches host-plant infection processes. Mol Biol Evol. 1993 Nov;10(6):1303–1316. doi: 10.1093/oxfordjournals.molbev.a040077. [DOI] [PubMed] [Google Scholar]
  4. Goodfellow M., Pirouz T. Numerical classification of sporoactinomycetes containing meso-diaminopimelic acid in the cell wall. J Gen Microbiol. 1982 Mar;128(3):503–527. doi: 10.1099/00221287-128-3-503. [DOI] [PubMed] [Google Scholar]
  5. Major J. G., Jr A rapid PCR method of screening for small mutations. Biotechniques. 1992 Jan;12(1):40–44. [PubMed] [Google Scholar]
  6. Nazaret S., Cournoyer B., Normand P., Simonet P. Phylogenetic relationships among Frankia genomic species determined by use of amplified 16S rDNA sequences. J Bacteriol. 1991 Jul;173(13):4072–4078. doi: 10.1128/jb.173.13.4072-4078.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Normand P., Cournoyer B., Simonet P., Nazaret S. Analysis of a ribosomal RNA operon in the actinomycete Frankia. Gene. 1992 Feb 1;111(1):119–124. doi: 10.1016/0378-1119(92)90612-s. [DOI] [PubMed] [Google Scholar]
  8. Normand P., Simonet P., Bardin R. Conservation of nif sequences in Frankia. Mol Gen Genet. 1988 Aug;213(2-3):238–246. doi: 10.1007/BF00339587. [DOI] [PubMed] [Google Scholar]
  9. Pernodet J. L., Boccard F., Alegre M. T., Gagnat J., Guérineau M. Organization and nucleotide sequence analysis of a ribosomal RNA gene cluster from Streptomyces ambofaciens. Gene. 1989 Jun 30;79(1):33–46. doi: 10.1016/0378-1119(89)90090-5. [DOI] [PubMed] [Google Scholar]
  10. Picard C., Ponsonnet C., Paget E., Nesme X., Simonet P. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Appl Environ Microbiol. 1992 Sep;58(9):2717–2722. doi: 10.1128/aem.58.9.2717-2722.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  12. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Simonet P., Grosjean M. C., Misra A. K., Nazaret S., Cournoyer B., Normand P. Frankia genus-specific characterization by polymerase chain reaction. Appl Environ Microbiol. 1991 Nov;57(11):3278–3286. doi: 10.1128/aem.57.11.3278-3286.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Simonet P., Le N. T., Du Cros E. T., Bardin R. Identification of frankia strains by direct DNA hybridization of crushed nodules. Appl Environ Microbiol. 1988 Oct;54(10):2500–2503. doi: 10.1128/aem.54.10.2500-2503.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Simonet P., Normand P., Moiroud A., Bardin R. Identification of Frankia strains in nodules by hybridization of polymerase chain reaction products with strain-specific oligonucleotide probes. Arch Microbiol. 1990;153(3):235–240. doi: 10.1007/BF00249074. [DOI] [PubMed] [Google Scholar]
  16. Stephen D., Jones C., Schofield J. P. A rapid method for isolating high quality plasmid DNA suitable for DNA sequencing. Nucleic Acids Res. 1990 Dec 25;18(24):7463–7464. doi: 10.1093/nar/18.24.7463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Tautz D., Renz M. An optimized freeze-squeeze method for the recovery of DNA fragments from agarose gels. Anal Biochem. 1983 Jul 1;132(1):14–19. doi: 10.1016/0003-2697(83)90419-0. [DOI] [PubMed] [Google Scholar]
  18. Winship P. R. An improved method for directly sequencing PCR amplified material using dimethyl sulphoxide. Nucleic Acids Res. 1989 Feb 11;17(3):1266–1266. doi: 10.1093/nar/17.3.1266. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES